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Objective To observe the effect of exosomes secreted by retinal pigment epithelial (RPE) cells which damaged by blue light to Nod-like receptor protein (NLRP3). Methods Cultured ARPE-19 cells were divided into 2 groups; one group of RPE cells were exposed to blue light irradiation for 6 hours, the other group was cultured in routine environment. Total exosomes were extracted from the two groups by differential ultracentrifugation in low-temperature, and examined by transmission electron microscope to identify their forms. The exosomes were then incubated with normal ARPE-19 cells. The expression level of CD63, interleukin (IL)-1β, IL-18 and caspase-1 on the exosome surface were measured by Western blotting. The expressions of NLRP3 mRNA in RPE cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR). Results Blue light damaged the cellular morphology. Transmission electron microscopy showed that the exosomes were 50-200nm in diameter and like double-concave disks. Blue light damaged cell-derived exosomes had significantly higher expression of IL-1β (t=18.04),IL-18 (t=12.55) and caspase-1 (t=14.70) than the control group (P<0.001). ARPE-19 cells cultured with blue light damaged cell-derived exosomes also had significantly higher expression of IL-1β (t=18.59), IL-18 (t=23.95) and caspase-1 (t=35.27) than control exosomes (P<0.001). RT-PCR showed that the relative expression of NLRP3 mRNA of PRE cells in experimental group and control group were 1.000±0.069 and 0.2±0.01, respectively, the difference was significant (t=12.20, P<0.001). Conclusion The expression IL-1β, IL-18 and caspase-1 and NLRP3 mRNA were upregulated by exosomes secreted by blue light damaged-RPE cells.
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Objective To observe the effect of exosomes secreted by retinal pigment epithelial (RPE) cells which damaged by blue light to Nod-like receptor protein (NLRP3). Methods Cultured ARPE-19 cells were divided into 2 groups; one group of RPE cells were exposed to blue light irradiation for 6 hours, the other group was cultured in routine environment. Total exosomes were extracted from the two groups by differential ultracentrifugation in low-temperature, and examined by transmission electron microscope to identify their forms. The exosomes were then incubated with normal ARPE-19 cells. The expression level of CD63, interleukin (IL)-1β, IL-18 and caspase-1 on the exosome surface were measured by Western blotting. The expressions of NLRP3 mRNA in RPE cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR). Results Blue light damaged the cellular morphology. Transmission electron microscopy showed that the exosomes were 50-200nm in diameter and like double-concave disks. Blue light damaged cell-derived exosomes had significantly higher expression of IL-1β (t=18.04),IL-18 (t=12.55) and caspase-1 (t=14.70) than the control group (P<0.001). ARPE-19 cells cultured with blue light damaged cell-derived exosomes also had significantly higher expression of IL-1β (t=18.59), IL-18 (t=23.95) and caspase-1 (t=35.27) than control exosomes (P<0.001). RT-PCR showed that the relative expression of NLRP3 mRNA of PRE cells in experimental group and control group were 1.000±0.069 and 0.2±0.01, respectively, the difference was significant (t=12.20, P<0.001). Conclusion The expression IL-1β, IL-18 and caspase-1 and NLRP3 mRNA were upregulated by exosomes secreted by blue light damaged-RPE cells.
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Objective To observe the influence of down-regulation of HtrA1 expression by small interfering RNA on light-injured human retinal pigment epithelium (RPE) cells.Methods Cultured human RPE cells(8th-12th generations)were exposed to the blue light at the intensity of (2000 ± 500) Lux for 6 hours to establish the light injured model.Light injured cells were divided into HtrA1 siRNA group,negative control group and blank control group.HtrA1 siRNA group and negative control group were transfected with HtrA1 siRNA and control siRNA respectively.The proliferation of cells was assayed by CCK-8 method.Transwell test was used to detect the invasion ability of these three groups.Flow cytometry was used to detect the cell cycle and apoptosis.The expression of HtrA1 and vascular endothelial growth factor (VEGF)-A was detected by real time-polymerase chain reaction and Western blot respectively.Results The mRNA and protein level of HtrA1 in the light injured cells increased significantly compared to that in normal RPE cells (t=17.62,15.09;P＜0.05).Compared with negative control group and blank control group,the knockdown of HtrA1 in HtrA1 siRNA group was associated with reduced cellular proliferation (t=6.37,4.52),migration (t =9.56,12.13),apoptosis (t =23.37,29.08) and decreased mRNA (t=17.36,11.32,7.29,4.05) and protein levels (t=12.02,15.28,4.98,6.24) of HtrA1 and VEGF-A.Cells of HtrA1 siRNA group mainly remained in G0/G1 phase,the difference was statistically significant (t=6.24,4.93;P ＜0.05).Conclusion Knockdown of HtrA1 gene may reduce the proliferation,migration capability and apoptosis of light-injured RPE cells,and decrease the expression of VEGF-A.
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Objective To investigate the effects of FTY720 on retinal photoreceptor cells and microglial following light-induced degeneration in rat retina.Methods 120 Sprague-Dawley rats were randomly divided into four groups including FTY720 group,solvent control group,model group and normal group.The rats of normal group were not intervened.The FTY720 group,solvent control group and model group establish retinal light injury mode.FTY720 was injected into abdominal cavity of the rats in FTY720 group 0.5 hours before light exposure.50％ dimethylsulfoxide was injected into abdominal cavity of the rats in solvent control group.The expressions of microglial cells in rat retinal were quantified using flow cytometry,the expressions of interleukin (IL)-1β were examined by enzyme-linked immuno sorbent assay at 6 hours,1 day,3 days,7 days after light exposure.The apoptosis of retinal photoreceptor cells were measured by terminal-deoxynucleoitidyl transferase mediated nick end labeling at 1 day after light exposure.The morphological change of retinal were viewed by haematoxylin and eosin staining at 7 days after light exposure.Results The expressions of microgilal and IL-1β began to rise at 1 day after light exposure,reached at peak at 3 days and decreased at 7 days.The expressions of IL-1βand microglial in FTY720 group were significantly lower than solvent control group and model group,but higher than normal group (P＜0.05).One day after exposure to light,the apoptosis cell ratio in normal group,model group,solvent control group and FTY720 group were 0,(87.66 ± 2.50) ％,(86.00 ± 2.44) ％,(49.66 ± 2.80) ％.The apoptosis cell in FTY720 group were higher than normal group,lower than solvent control group and model group (P＜0.05).Seven days after exposure to light,the retinal in normal group was structured and the cell was arranged well,the cell in solvent control group and model group was irregular arrangement and the outer nuclear layer (ONL) was thin after light exposure.The thickness of the ONL in FTY720 group was significantly higher than solvent control group and model group,below normal group.Conclusion FTY720 can prevents retinal photoreceptor cells from apoptosis and inhibits activation of microglial.
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Objective To assess the effects of 670nm LED (light-emitting diode) to protect the photoreceptor from the light-induced damage in a rat model. Methods 32 SD rats were randomly assigned to one of eight groups: untreated control group, the LED-treated control group, three groups of light-induced damage,and three groups of light-induced damage treated with LED. Light-induced damage result from exposing to constant light for 3 hours of different illuminations of 900,1800 and 2700 lx, respectively. The LED treatment (50 mW) was delivered for 30 minutes at 3 hours before the light damage and 0,24 and 48 hours after the light damage. Retinal function and morphology were measured by electroretinogram (ERG) and histopathology assay. Results The illumination of 900 lx for 3 hours did not damage the rat retina. The illumination of 1800 lx for 3 hours resulted in thinner ONL and no OS and IS. The ratio of damaged area/total retinal area was 0.48±0.12, the damaged thickness of ONL/normal ONL (L5) was 0.39±0.07,and the amplitude of ERG b wave was (431±120) μV. With the LED treatment the ratio of damaged area decreased (M6=0.17±0.12, P5/6=0.002), and the ratio of the damaged thickness of ONL also decreased (L6=0.22±0.09, P5/6<0.01), and the amplitude of ERG b wave increased to (1011±83) μV(P5/6 <0.001). The illumination of 2700 lx for 3 hours caused severed damage to the rat retina and the LED could not protect them significantly. Conclusions 670 nm LED treatment has an evident protective effect on retinal cells against light-induced damage, which may be a simple and effective therapy to prevent or to delay age-related maeular degeneration.